Several references and patents are cited throughout the present application in order to more clearly describe the state of the art to which the invention pertains. Each of the above described citations is incorporated by reference herein.
In conventional (non-enzymatic) chemical synthesis processes, esterification of compounds with multiple hydroxyl groups (e.g., polyalcohols such as glycerol), will, in general, yield a product mixture of mono- and poly-substituted esters, rather than a product with predominantly only one particular hydroxyl group esterified. The esterification can be made selective by adding protecting groups prior to the esterification step. The protecting groups are then normally removed after the esterification. The use of such protecting groups complicates and adds expense to the synthesis. Similarly, synthesis routes involving activated carboxylic acid derivatives form undesired secondary products. The formation of unwanted secondary products reduces the yield of the desired products, burdens subsequent purification steps, and may create additional waste disposal issues.
Another class of reactions prevalent in the processing of fats include transesterifications, re-esterifications, and acyltransfers. Enzymatic processes for esterification or reesterification and acyltransfer are well known in the art. For instance,it is well established that transesterification can be catalyzed by lipases in water-free media. If water is present in the reaction of esters, alcohol and lipases, cleavage of the acids and/or intermediates thereof normally occurs. Since various lipases also catalyze the formation of esters from free fatty acids and alcohols, lipase catalyzed transesterifications normally proceed through an acid intermediate stage. However, in many commercial processes, the presence of free acids is undesirable. The water content prevents, to some extent, a technically and commercially acceptable reaction (formation of an unfavorable thermodynamic equilibrium). Thus, costly operations including water removal by such methods as azeotropic distillation, membrane separation processes, vacuum distillation have been employed in order to achieve satisfactory yields.
The above mentioned difficulties can be avoided, or at least partly ameliorated, by employing enzyme catalysts. The use of enzyme catalysts 1) permits milder reaction conditions, 2) enhances the selectivity of the synthesis, 3) reduces unwanted secondary products, 4) results in a less expensive synthetic process and 5) facilitates post-synthesis purification.
In light of the above, enzyme catalysis is finding increasing and wider application in chemical and biochemical synthesis. More specifically, and more germane to the present invention, hydrolases and especially lipases (two classes of proteins with enzyme activity for breaking down molecules) are employed for splitting fat compounds (e.g., triacyl glycerides) in large-scale industrial processes. It is an object of the invention to provide improved lipases/acyltransferases and nucleic acids encoding the same, for this and a variety of other purposes.